A novel strategy for the discovery of MHC class II-restricted tumor antigens: identification of a melanotransferrin helper T-cell epitope

CD4+ helper T cells play a critical role in orchestrating host immune responses, including antitumor immunity. The limited availability of MHC class II-associated tumor antigens is still viewed as a major obstacle in the use of CD4+ T cells in cancer vaccines. Here, we describe a novel approach for the identification of MHC class II tumor-associated antigens (TAAs). By combining two-dimensional liquid chromatography and nanoelectrospray ionization tandem mass spectrometry, we developed a highly sensitive method for the detection of human leukocyte antigen (HLA)-DR-associated peptides of dendritic cells upon exposure to necrotic tumor cells. This approach led to the identification of a novel MHC class II-restricted TAA epitope derived from melanotransferrin. The epitope stimulated T cells derived from melanoma patients and healthy individuals and displayed promiscuity in HLA-DR restriction. Moreover, the same peptide was also presented by MHC class II-positive melanoma cells. This strategy may contribute to increase the number of tumor epitopes presented by MHC class II molecules and may support the development of more efficacious vaccines against cancer.     

Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informativemodel for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction.     

Identification of a tumor-specific shared antigen derived from an Eph receptor and presented to CD4 T cells on HLA class II molecules

We obtained a lytic CD4 T-cell clone that recognized an antigen presented by HLA-DRB1*1101 on the tumor cells of a melanoma patient who enjoyed an unusually favorable clinical evolution. The antigen appeared to be shared between several melanoma cell lines. To identify the encoding gene, we used a new method, based on the cotransfection into human embryonal kidney cell line 293 of a cDNA library from the tumor together with a cDNA clone encoding the class II transactivator, which induces the expression of HLA class II molecules. The product of the gene coding for the antigenic peptide is EphA3, a member of the Eph family of tyrosine kinase receptors, which mediate the repulsion of neural cells by cells carrying the ligand Ephrins on their surface. EphA3 is expressed at a high level in the retina and fetal brain, at a lower level in several normal tissues, and not at all in hematopoietic cells, the only cells that constitutively express HLA class II molecules. It is overexpressed in several types of tumors, including melanoma, lung carcinoma, and sarcoma. On the basis of this pattern of expression, EphA3 may be a source of tumor-specific antigens recognized on tumor cells that express HLA class II molecules. Anti-EphA3 T cells may have participated in a tumor rejection response in the patient, because the cells of metastases collected several years later than the metastasis used to characterize the antigen had lost expression of HLA-DR or EphA3, therefore escaping recognition by these lymphocytes.     

Major histocompatibility antigens and antigen-processing molecules in uveal melanoma.

PURPOSE: Malignant transformation of cells is frequently associated with abnormalities in the human leukocyte antigen (HLA) expression. These abnormalities may play a role in the clinical course of the disease, because HLA antigens mediate interactions of tumor cells with T cells and natural killer cells. Uveal melanoma is a highly malignant tumor of the eye and is characterized by hematogenic spread to liver. Antigen-processing molecules (APMs) are necessary for efficient expression of HLA class I antigens. We studied the expression of HLA antigens and the APM in uveal melanomas by immunohistochemistry and correlated clinicopathologically. EXPERIMENTAL DESIGN: HLA class I antigen, beta(2)-microglobulin (beta(2)-m), HLA class II antigens, and the APM comprising proteasomal subunits low molecular mass polypeptide (LMP) 2, beta-subunit of LMP2-Delta, LMP 10, transporter associated protein 1 subunit, and chaperone molecules tapasin and calnexin were studied in 41 primary uveal melanoma archival specimens by immunohistochemistry. Immunoanalysis was done by a semiquantitative method and correlated with extrascleral extension, cell types, and the largest tumor diameter. RESULTS: HLA class I antigen, beta(2)-m, HLA class II antigen, and the APM were decreased (negative staining in 29 tumors and dull staining in 3 tumors) in 100% (32 of 32) uveal melanomas with no extrascleral extension. (P = 0.01) and positive (bright staining) in 67% (4 of 9) tumors with liver metastasis. Decreased immunoexpression of HLA antigens and the APM was seen in nonepithelioid cell melanomas. There was no correlation with largest tumor diameter. CONCLUSIONS: Our data suggest decreased expression of HLA, and APM are seen in uveal melanomas with no extrascleral extension and in nonepithelioid cell melanomas. Decreased expression of APM may contribute to decreased HLA class I antigen expression.     

Cancer immunotherapy using novel tumor‐associated antigenic peptides identified by genome‐wide cDNA microarray analyses

Recent genome‐wide cDNA microarray analysis of gene expression profiles in comprehensive tumor types coupled with isolation of cancer tissues by laser‐microbeam microdissection have revealed ideal tumor‐associated antigens (TAAs) that are frequently overexpressed in various cancers including head and neck squamous cell cancer (HNSCC) and lung cancer, but not in most normal tissues except for testis, placenta, and fetal organs. Preclinical studies using HLA‐transgenic mice and human T cells in vitro showed that TAA‐derived CTL‐epitope short peptides (SPs) are highly immunogenic and induce HLA‐A2 or ‐A24‐restricted CTLs. Based on the accumulated evidence, we carried out a phase II clinical trial of the TAA‐SP vaccine in advanced 37 HNSCC patients. This study showed a significant induction of TAA‐specific CTLs in the majority of patients without serious adverse effects. Importantly, clinical responses including a complete response were observed in this study. Another phase II clinical trial of therapeutic TAA‐SP vaccine, designed to evaluate the ability of prevention of recurrence, is ongoing in HNSCC patients who have received curative operations. Further studies in human preclinical studies and in vivo studies using HLA class I transgenic mice showed TAA‐derived long peptides (TAA‐LPs) have the capacity to induce not only promiscuous HLA class II‐restricted CD4⁺ T helper type 1 cells but also tumor‐specific CTLs through a cross‐presentation mechanism. Moreover, we observed an augmentation of TAA‐LP‐specific T helper type 1 cell responses and tumor antigen‐spreading in HNSCC patients vaccinated with TAA‐SPs. This accumulated evidence suggests that therapeutic TAA‐SPs and LPs vaccines may provide a promising cancer immunotherapy.     

Influence of major histocompatibility complex class I, class II and TLA genes on tumor rejection.

T lymphocytes recognize antigen associated with MHC class I and/or class II gene products. Recognition of malignant cells is therefore dependent on presentation of tumor associated antigen(s) by MHC molecules. We have studied immunity to tumors that have down-regulated class I expression. These studies demonstrate a requirement for class I antigens, but suggest that additional factors may also be required for tumor-specific immunity. The MHC also encodes TLA class I antigens, whose function is unknown. Our studies suggest that these molecules function is unknown. Our studies suggest that T lymphocytes, specifically in tumor cells that do not express H-2K or H-2D moieties. Other studies are aimed at improving tumor-specific Th cell generation by producing class II+ tumor cells. The success of these experiments indicates that this approach may be a potentially useful immunotherapy.     

Molecular mechanisms of HLA class I antigen abnormalities following viral infection and transformation

In humans as in other animal species, CD8+ cytotoxic T lymphocytes (CTLs) play an important if not the major role in controlling virus-infected and malignant cell growth. The interactions between CD8+ T cells and target cells are mediated by human leukocyte antigen (HLA) class I antigens loaded with viral and tumor antigen-derived peptides along with costimulatory receptor/ligand stimuli. Thus, to escape from CD8+ T-cell recognition and destruction, viruses and tumor cells have developed strategies to inhibit the expression and/or function of HLA class I antigens. In contrast, cells with downregulated MHC class I surface expression can be recognized by NK cells, although NK cell-mediated lysis could be abrogated by the expression of inhibiting NK cell receptors. This review discusses the molecular mechanisms utilized by viruses to inhibit the formation, transport and/or expression of HLA class I antigen/peptide complexes on the cell surface. The knowledge about viral interference with MHC class I antigen presentation is not only crucial to understand the pathogenesis of viral diseases, but contributes also to the design of novel strategies to counteract the escape mechanisms utilized by viruses. These investigations may eventually lead to the development of effective immunotherapies to control viral infections and virus-associated malignant diseases.     

Loss of HLA haplotype in lung cancer cell lines: implications for immunosurveillance of altered HLA class I/II phenotypes in lung cancer.

Loss of expression of HLA class I antigens has been demonstrated in a wide variety of tumors and is considered to be one of the mechanisms whereby tumors escape T-cell surveillance. Genomic DNA of MHC class I/II molecules in seven lung cancer cell lines was investigated and compared with that in peripheral blood mononuclear cells. In three cell lines, OU-LC-A1, OU-LC-A2, and OU-LC-AS1, a loss of HLA haplotype was observed. Aberrations of HLA class I/II in tumor cell lines should be considered when MHC-restricted phenomena in vitro models are assessed and clinical use of tumor vaccination in vivo is considered.     

Strategies to endow cytotoxic T lymphocytes or natural killer cells with antibody activity against carcinoembryonic antigen.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are main effecter cells in cellular immunity against tumor cells. T-cell immunotherapy is based on the assumption that tumor(-associated) antigen (TA) peptides are correctly presented by HLA class I molecules on target tumor cells, and NK cell immunotherapy is based on the hypothesis that cell surface TAs or ligands for NK receptors are widely expressed in tumor cells. However, human tumor cells often lose HLA class I molecules, and target cell ligands for NK receptors are not always expressed in human tumor cells. These altered HLA class I phenotypes and non-ubiquitous expression of NK receptor ligands constitute the major tumor escape mechanism facing tumor-specific CTL and/or NK cell mediated responses. These facts also indicate that it is not easy to eliminate the target tumors only by activating tumor-specific CTLs or NK cells with cancer vaccine treatments. On the other hand, it is easily confirmed by immunohistochemistry whether or not antibody-recognized TAs exist on the cell surface of target tumor cells. Therefore, endowing CTLs or NK cells with antigen-binding specificity of anti-TA antibody is a promising approach for re-targeting the activities of these effector cells to tumor cells in an HLA-independent manner. This review summarizes the following four new strategies for re-targeting CTLs or NK cells to carcinoembryonic-antigen-expressing tumor cells: (1) bispecific antibody technology; (2) antibody-cytokine fusion protein technology; (3) chimeric immune receptor technology, and (4) antibody-HLA/peptide complex technology.     

T-cell receptor transgenic analysis of tumor-specific CD8 and CD4 responses in the eradication of solid tumors

The role of tumor-specific CD8 and CD4 lymphocytes in rejecting solid tumors has been difficult to determine because of the lack of models in which tumor antigen, specific CD8 cells, and specific CD4 cells can be monitored and controlled. To investigate the minimal components required for the induction and maintenance of CTL activity sufficient to reject a solid tumor in vivo, we transfected the influenza hemagglutinin (HA) gene into a nonimmunogenic class I+/class II- murine malignant mesothelioma (MM) tumor line to generate an endogenous tumor antigen and used TCR transgenic mice with class I- or class II-restricted specificities for HA as sources of naive, tumor-specific T cells. The data show that the presence of a strong tumor antigen is not in itself sufficient to induce an effective CTL response, nor does the presence of a high frequency of precursor cells guarantee tumor rejection. We also show that tumor-specific CD4 cells, when CTL numbers are suboptimal, greatly enhance the eradication of tumor, confirming the importance of antigen-presenting cell presentation of tumor antigens to class II-restricted cells. These data confirm that T-cell receptor transgenic cells, combined with nominal tumor antigen transfection, represent powerful tools to analyze tumor-specific T-cell responses.     

Dendritic cells, pulsed with lysate of allogeneic tumor cells, are capable of stimulating MHC-restricted antigen-specific antitumor T cells

A variety of approaches have been used to deliver tumor-associated antigens (TAA) in conjunction with dendritic cells (DC) as cellular adjuvants. DC derived from monocytic precursors have been pulsed with whole tumor antigen using a variety of strategies and have been demonstrated to induce CD4+ and CD8+ antitumor responses. In the present study, monocyte-derived DC have been pulsed with lysate from an allogeneic melanoma cell line, A-375, and used to repeatedly stimulate T cells. The resultant T cells were examined for cytotoxic activity against A-375 targets as well as the HLA A2-positive melanoma cell line DFW. Uptake of FITC-labeled melanoma lysate by DC established that lysate of melanoma cells was efficiently endocytosed. Stimulation with lysate-pulsed DC resulted in strong proliferative responses by T cells, which could be inhibited by antibodies against both MHC class I and class II. T cells stimulated in vitro with lysate-pulsed DC demonstrated potent cytotoxicity against the melanoma targets which were blocked by antibodies against MHC class I. Lysate-pulsed DC also elicited IFN-gamma secretion by T cells as measured in an ELISPOT assay. We have also examined the ability of lysate-pulsed DC to present melanoma-associated antigens to T cells. ELISPOT assays with synthetic peptides of melanoma-associated antigens, such as gp100, mage1, NY-ESO, and MART-1, revealed that lysate-pulsed DC could stimulate T cells in an antigen-specific manner. The results demonstrate that lysate from allogeneic tumor cells may be used as a source of antigens to stimulate tumor-specific T cells in melanoma.     

Human leukocyte antigen and antigen processing machinery component defects in astrocytic tumors.

PURPOSE: To determine the frequency of abnormalities in human leukocyte antigen (HLA) and antigen processing machinery (APM) component expression in malignant brain tumors. This information may contribute to our understanding of the immune escape mechanisms used by malignant brain tumors because HLA antigens mediate interactions of tumor cells with the host's immune system. EXPERIMENTAL DESIGN: Eighty-eight surgically removed malignant astrocytic tumors, classified according to the WHO criteria, were stained in immunoperoxidase reactions with monoclonal antibody recognizing monomorphic, locus-specific, and allospecific determinants of HLA class I antigens, beta2-microglobulin, APM components (LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin), and HLA class II antigens. RESULTS: HLA class I antigens were lost in approximately 50% of the 47 glioblastoma multiforme (GBM) lesions and in approximately 20% of the 18 grade 2 astrocytoma lesions stained. Selective HLA-A2 antigen loss was observed in approximately 80% of the 24 GBM lesions and in approximately 50% of the 12 grade 2 astrocytoma lesions stained. HLA class I antigen loss was significantly (P < 0.025) correlated with tumor grade. Among the APM components investigated, tapasin expression was down-regulated in approximately 20% of the GBM lesions analyzed; it was associated, although not significantly, with HLA class I antigen down-regulation and tumor grade. HLA class II antigen expression was detected in approximately 30% of the 44 lesions analyzed. CONCLUSION: The presence of HLA antigen defects in malignant brain tumors may provide an explanation for the relatively poor clinical response rates observed in the majority of the T cell-based immunotherapy clinical trials conducted to date in patients with malignant brain tumors.     

CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells

Although they are considered as antigen-presenting cells, the role of antigen-unspecific B lymphocytes in antigen presentation and T-lymphocyte stimulation remains controversial. In this paper, we tested the capacity of normal human peripheral activated B cells to stimulate T cells using melanoma antigens or melanoma cell lysates. B lymphocytes activated through CD40 ligation and then pulsed with tumor antigens efficiently processed and presented MHC class II-restricted peptides to specific CD4(+) T-cell clones. This suggests that CD40-activated B cells have the functional and molecular competence to present MHC class II epitopes when pulsed with exogenous antigens, thereby making them a relevant source of antigen-presenting cells to generate T cells. To test this hypothesis, CD40-activated B cells were pulsed with a lysate prepared from melanoma cells and used to stimulate peripheral autologous T cells. Interestingly, T cells specific to melanoma antigens were generated. Additional analysis of these T-cell clones revealed that they recognized MHC class II-restricted epitopes from tyrosinase, a known melanoma tumor antigen. The efficient antigen presentation by antigen-unspecific activated B cells was correlated with a down-regulation in the expression of HLA-DO, a B cell-specific protein known to interfere with HLA-DM function. Because HLA-DM is important in MHC class II peptide loading, the observed decrease in HLA-DO may partially explain the enhanced antigen presentation after B-cell activation. Results globally suggest that when they are properly activated, antigen-unspecific B-lymphocytes can present exogenous antigens by MHC class II molecules and stimulate peripheral antigen-specific T cells. Antigen presentation by activated B cells could be exploited for immunotherapy by allowing the in vitro generation of T cells specific against antigens expressed by tumors or viruses.     

MHC antigens in human melanomas

The distribution of MHC antigens in human melanocytic lesions, i.e. HLA class I and HLA class II antigens is reviewed. HLA class I antigens have a broad distribution, but may be lost during tumor progression. In contrast, HLA class II antigen expression appears with neoplastic transformation. The mode of regulation of HLA antigens in melanoma lesions is complex. Immunohistochemical demonstration of HLA antigen expression in primary melanoma lesions and in locoregional metastases has prognostic relevance. Expression of HLA-DR in primary melanoma lesions is associated with an unfavorable prognosis, as is a decreased expression of HLA-A,B,C antigens in locoregional metastases.     

Gene expression profiling identifies BAX-delta as a novel tumor antigen in acute lymphoblastic leukemia

The identification of new tumor-associated antigens (TAA) is critical for the development of effective immunotherapeutic strategies, particularly in diseases like B-cell acute lymphoblastic leukemia (B-ALL), where few target epitopes are known. To accelerate the identification of novel TAA in B-ALL, we used a combination of expression profiling and reverse immunology. We compared gene expression profiles of primary B-ALL cells with their normal counterparts, B-cell precursors. Genes differentially expressed by B-ALL cells included many previously identified as TAA in other malignancies. Within this set of overexpressed genes, we focused on those that may be functionally important to the cancer cell. The apoptosis-related molecule, BAX, was highly correlated with the ALL class distinction. Therefore, we evaluated BAX and its isoforms as potential TAA. Peptides from the isoform BAX-delta bound with high affinity to HLA-A*0201 and HLA-DR1. CD8+ CTLs specific for BAX-delta epitopes or their heteroclitic peptides could be expanded from normal donors. BAX-delta-specific T cells lysed peptide-pulsed targets and BAX-delta-expressing leukemia cells in a MHC-restricted fashion. Moreover, primary B-ALL cells were recognized by BAX-delta-specific CTL, indicating that this antigen is naturally processed and presented by tumor cells. This study suggests that (a) BAX-delta may serve as a widely expressed TAA in B-ALL and (b) gene expression profiling can be a generalizable tool to identify immunologic targets for cancer immunotherapy.     

MHC class I and class II presentation of tumor antigen in retrovirally and adenovirally transduced dendritic cells

The unique antigen-presenting capabilities of dendritic cells (DCs) make them an attractive means with which to initiate an antitumor immune response. Using DCs transduced with tumor antigens for immunotherapy has several theoretical advantages over peptide-pulsed DCs including the possibility that transduced DCs are capable of presenting epitopes on both class I and class II MHC molecules. To test this theory, we inserted the human tumor antigen gp100 into mouse DCs transgenic for HLA-DRbeta1*0401 using either adenoviral vector or a VSV-G pseudotyped retroviral vector. DCs transduced with tumor antigen were able to be recognized by both a murine CD8(+) T-cell clone and a murine CD4(+) T-cell line in a cytokine release assay, thereby demonstrating presentation of both MHC class I and class II gp100 epitopes. This study describes the simultaneous presentation of a tumor-associated antigen to both CD4(+) and CD8(+) T cells and lends support to the use of gene-modified DCs as a means to initiate both CD4(+) and CD8(+) antitumor responses.     

Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs). Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid. Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE. While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-α) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185^HER-2/neu were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MAGE, GAGE, EGFR, p185^HER-2/neu and FR-α expressed in INT.Ov lines. Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide-based vaccines. Int. J. Cancer 73:143–150, 1997. © 1997 Wiley-Liss, Inc.     

Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLA-A2.

Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.     

An immunohistochemical study of altered immunomodulatory molecule expression in head and neck squamous cell carcinoma.

For the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system, the expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary. There is increasing evidence that surface HLA class I antigen expression is altered in a variety of human tumours by either loss or down-regulation of these molecules, which may be a strategy for evasion of immunosurveillance by malignant cells. This study has examined the expression of HLA class I molecules in head and neck squamous cell carcinoma (HNSCC) specimens by immunohistochemistry, using a wide panel of antibodies directed against allele-specific as well as monomorphic determinants of these molecules. The expression of TAP proteins, HLA-DR and the co-stimulatory molecule ICAM-1 were also studied. In addition, the expression of the tumour-associated antigens (TAA) p53 and MAGE genes was determined. Aberrant allelic expression of HLA class I antigens was detected in 17 out of 34 (50%) of the specimens stained, whereas HLA class I expression determined by W6/32 staining was found to be heterogeneous in only 2 out of 34 (6%) cases. Decreased expression of ICAM-1 was observed in 12 out of 34 (35%) tumour specimens and de novo expression of HLA-DR (HLA class II) by carcinoma cells in 13 out of 34 (38%) cases. Aberrant expression of HLA class I antigens was frequently observed in cases in which MAGE genes and p53 overexpression were detected. The altered expression of these immunomodulatory molecules in HNSCC may affect prognosis and has important implications for peptide-based immunotherapy strategies for these patients.